Proper vascular modeling in the retina is essential for ocular development and visual acuity. Abnormal vessel growth during development or in adulthood produces several relatively common diseases such as retinopathy of prematurity, diabetic retinopathy, and age-related macular degeneration. Normal retinal development occurs through vessels forming at the optic nerve head and spreading over the retina to form a dense network. Connolly, S E, et al, Microvasc Res, 1988; 36:275-290; Provis, J M, Prog Retin Eye Res, 2001; 20:799-821; Fruttiger, M, Invest Ophthalmol Vis Sci, 2002; 43:522-527. Development proceeds through formation of primary vessels along the surface of the developing retina from which divergent vessels begin to extend into the capillary beds that form the outer and inner plexiform layers of the retina. Connelly, 1988; Provis, 2001, Fruttiger, 2002. Vascular development is mediated by a series of growth factors that direct formation and extension of new vessels. Retinal development is unique in the concentration and types of signaling mediators employed to promote angiogenic sprouting from the primary vascular network and the formation of the final capillary architecture. Ohlmann, A, et al, J Neurosci, 2005; 25:1701-1710. One factor hypothesized to be involved in formation of primary retinal vasculature and retinal capillaries is the protein Norrin. Norrin, is a 131 amino acid long protein that is secreted into the extracellular space. Meitinger, T, et al, Nat Genet, 1993; 5:376-380; Berger, W, et al, Hum Mol Genet, 1996; 5:51-59. Two primary domains define the general Norrin protein structure: a signal peptide directs localization of the molecule; and a cysteine-knot motif provides the tertiary confirmation required for receptor binding and activation of signal transduction.
The importance of the cysteine knot-motif is highlighted by computer modeling that demonstrates the requirement of disulfide bonds between the cysteine residues in forming the structural confirmation of Norrin. Mutation(s) of the cysteine residues reduces the affinity of Norrin for its receptor and prevents activation of subsequent signaling pathways. Mutations in these residues also result in severe retinal dysgenesis and Norrie disease. However, mutations in regions other than the cysteine knot-motif produce incomplete protein folding and result in familial exudative vitreoretinopathy (FEVR) and related vitreoretinopathies (Retinopathy of Prematurity, persistent fetal vasculature).
Norrin is a ligand for the Frizzled receptor subtype 4 (Fz4). Norrin binds Fz4 with nanomolar affinity and stimulates a Wnt receptor:β-catenin signal transduction pathway that regulates retinal development and is necessary for regression of hyaloid vessels in the eye. Xu, Q, et al, Cell, 2004; 116:883-895; Clevers, H, Curr Biol, 2004; 14:R436-437; Nichrs, C, Dev Cell, 2004; 6:453-454. Norrin interaction with Fz4 is dependent on the cell surface receptor LRP5. Xu, 2004. Frizzled receptors are coupled to the β-catenin canonical signaling pathway that functions by the activation of Wnt target genes. Wnt protein binding to Frizzled and LRP5 inactivates glycogen synthase kinase (GSK) 3β and Axin. The inactivation of these proteins stabilizes β-catenin, which subsequently accumulates in the cell nucleus and activates the transduction of target genes that are crucial in the G1-S-phase transition, such as cyclin D1 or c-Myc. Willert K, and Nusse R, Curr Opin Genet Dev, 1998: 8:95-102. These pathways promote stimulation and proliferation of retinal stem cells. Inoue, T, et al, Stein Cells, 2006; 24:95-104.
Norrin is encoded by the NDP gene present on chromosome X at position 11.4. The importance of this gene product is highlighted by observations that inactivating mutations lead to Norrie disease which is characterized by ocular and cochlear vascular defects. Rhem, H L, et al, J Neurosci, 2002; 22:4286-4292; Black, G C, et al, Hum Mol Genet, 1999; 8:2031-2035. Silencing of the NDP gene produces incomplete regression of the primary hyaloid system and abnormal retinal maturation.
Observations that abnormalities in the Fz4 and LRP5 receptors that result in the phenotypically similar condition, FEVR underscore the importance of Norrin signaling. Robitaille, J, et al, Nature Genet, 2002; 32:326-330; Kondo, H, et al, Br J Opthalnol, 2003; 87:1291-1295; Toomes, C, et al, Am J Hum Genet, 2004; 74:721-730. The close association between the phenotypes produced by Norrin mutations and mutations in the Fz4 and LRP5 receptors bolsters the hypothesis that these molecules form a functional signaling group. Planutis, K, et al, BMC Cell Biology, 2007; 8:12.
While defects in the NDP gene and diseases due to incomplete or immature vascularization have been studied and correlated with disease, therapies presently available for Norrie disease, FEVR, or other retinal diseases are only modestly effective. Thus, there exists a need for improved therapeutics and methods of treatment fbr vitreoretinal disease and vascular disease in the retina.